Particle radiotherapy programs (pions and heavy ions) have reached the stage of treating specific tumor sites. For pion therapy, these sites are the lower abdomen (bladder, prostate and rectum) and the brain. With the anticipated longer survival times, the risk of developing late radiation damage in these organs increases. Therapists can not easily change the fractionation schemes because of a fear of such late effects. However, altered fractionation schemes may be required for particle therapy and such changes will be aided by the proposed preclinical experiments. In order to optimize treatment schemes for different sites, quantitative information is required on normal tissue tolerance to late-expressed damage at clinically relevant doses per fraction. The proposed project is aimed at obtaining critical biological data to support particle radiotherapy programs. The specific aims are: 1) to study the tolerance to late-expressed damage of the central nervous system, lung, bladder, and rectum; 2) to measure the RBE for acute effects on the small intestine using the crypt cell survival assay; and 3) to carry out in vitro studies to biologically characterize different pion beams using cultured cells and spheroids. The late effect studies are aimed at evaluating the relative importance of sublethal damage repair (by varying fraction size) and repopulation (by varying overall treatment times) in each tissue. The endpoints to be used will primarily involve non-invasive techniques that allow longitudinal measurement of organ function. In parallel to the functional assays, histological studies will be done to characterize different phases of damage and to identify possible target cells. The in vitro experiments will provide data concerning the effects of different pion beam stopping volumes on the relative biological effectiveness in a cultured cell system. Since all these experiments involve x-ray controls, our experiments are also aimed at improving understanding of conventional x-ray responses.